Many genes for heart-restricted transcription factors are regulated by modular enhancers that function at particular stages or in distinct regions of the developing heart. We propose to analyze and exploit two such heart-specific enhancers from the cGATA-6 gene that function in transgenic mice and serve as "biosensors" for several poorly understood facets of heart development. One enhancer is activated at the outset of the cardiogenic program and is silenced as the heart matures into a multichambered organ. The other enhancer selectively functions in myocardial cells that initiate atrioventricular valve formation and, at a later stage, in cells that comprise atrioventricular components of the cardiac conduction system. This heart-region-specific enhancer provides a novel means to selectively express or delete genes in these biomedically relevant subsets of myocardial cells and to evaluate the functional consequences of such precise genetic manipulations. This targeted approach provides a valuable complement to traditional gene knockout approaches which often result in early embryonic lethality or complex heart disorders. Another long-term goal is to understand how cardiac enhancers integrate diverse signals within chromosomal contexts and convert these into the transcriptional outputs that govern heart development. We propose to use a combination of in vivo footprinting and transgenic assays to achieve this goal. This approach has the potential to provide mechanistic insights about known heart-restricted factors and to reveal roles for novel transcription factors. Moreover, it is possible to ascertain whether binding site mutations also cause in vivo footprints to be perturbed over the rest of the enhancer. Indeed, this approach has provided evidence for novel factors that are required to activate and silence the early-heart-specific enhancer at different stages of heart development and it is interesting to note that this silencing element maps near binding sites for several evolutionarily conserved heart-restricted transcription factors. We will also attempt to use adjunct cell culture systems to facilitate our analysis of this early-heart-specific enhancer as well as the heart-region-specific enhancer described above.